The intestinal microbiota has been identified as a significant environmental factor regulating systemic neutrophil function. What we don't understand are the specific aspects of systemic neutrophil function that are stimulated by the microbiota, or the host signal transduction mechanisms that control these neutrophil responses. These gaps in our knowledge are important, because without this information, the development of methods for increasing or decreasing neutrophil activity by modulating host responses to the microbiota, or by altering the microbiota itself, is highly unlikely. Our long-term goal is to understand the molecular mechanisms underlying host-microbe interactions in the vertebrate intestine, and how those interactions systemically impact upon host health and disease. We have developed a gnotobiotic zebrafish system that facilitates rapid genetic tests and high-resolution imaging of immune cells in live animals. We have used this zebrafish model to reveal evolutionarily conserved roles for the microbiota on systemic neutrophil function, and novel systemic inflammatory roles for zebrafish serum amyloid A (Saa). The overall objective of this application is to exploit the advantages of the zebrafish system to define the molecular and cellular mechanisms by which the microbiota regulates systemic neutrophil development and function. The proposed research will address the central hvpothesis that distinct aspects of systemic neutrophil function are stimulated by the gut microbiota, and regulated by tissue-specific transcription and signaling of Saa. The rationale that underlies this research is that the identification of mechanisms that control systemic neutrophil function will provide new therapeutic strategies for treating inflammatory diseases such as inflammatory bowel diseases and spondyloarthritis, and reducing secondary infections in patients treated with broad-spectrum antibiotics, by controlling neutrophil production, localization, or activity. We plan to achieve the objective of this proposal through two specific aims. Under the first aim, we will define the precise roles of the gut microbiota on systemic neutrophil development and function using a photoconvertible fluorescent protein labeling method to track neutrophil dynamics in live gnotobiotic zebrafish. In the second aim, we will use genetic and transgenic analysis to define the mechanisms mediating Saa-dependent regulation of neutrophil responses to the microbiota. The proposed research is innovative because it constitutes the first analysis of commensal microbial regulation of systemic leukocyte biology in a gnotobiotic vertebrate host where all tissues can be directly viewed in live animals. Additionally, this is the first in vivo investigation of the transcriptional regulation and structure-function relationships of the Saa. The contribution of the proposed research will be significant because it will provide a much-needed vertical advance in our understanding of the gut microbiota's roles in systemic innate immunity, which is expected to lead to new probiotic, antibiotic, and pharmacologic strategies for controlling systemic leukocyte function to promote human health.